Brain derived neurotrophic factor (BDNF) stimulates the growth and survival of neuronal cells. Mild reductions in BDNF are associated with normal aging, memory impairment, neuropsychiatric and neurodegenerative disorders. Increased BDNF levels have been demonstrated to lift depression, prevent cell death and stimulate neuronal growth. In humans, a single nucleotide polymorphism in BDNF generates a valine to methionine substitution at residue 66 (Val66Met), located in the BDNF prodomain. The Met66 prodomain isoform is associated with memory impairment, depression and elevated risk of anxiety disorders. This substitution is found in 20% of the population, with 4% being homozygous. Common antidepressant therapies, that generally elevate BDNF levels, lose efficacy in patients bearing this isoform. We have recently observed that mature BDNF binds to its prodomain through interactions that directly involve the critical Val66Met site. In general, prodomain regions play important roles in protein folding, trafficking, stabilization, storage and regulation of mature growth factors. Specific isoform related differences in prodomain-BDNF interactions could provide a structural mechanism for the Met66 prodomain isoforms degenerative activity. We find that the isolated prodomain binds to mature BDNF with an affinity that suggests the complex could remain intact following secretion, potentially influencing BDNF activity. The goal of the proposed research is to structurally characterize this complex and elucidate critical differences in the Val66 and Met66 prodomain interactions with BDNF. To accomplish this goal we will pursue the following specific aims. Specific Aim 1: Produce and structurally characterize prodomain isoforms and mature BDNF growth factor. Specific Aim 2: Map the BDNF-prodomain binding interface and characterize differences in stability and function. We will pursue these aims using a combination of NMR and biophysical techniques combined with functional assessment of bioactivity using cell based assays. Many of the techniques applied in this study have not been directed toward the study of BDNF or other neurotrophin growth factors. We anticipate that this project will identify new molecular details about prodomain isoform interactions responsible for Val66Met differences in BDNF activity and function.